This invention comprises the transgenic soybean (Glycine max) plant MON87754 with elevated seed oil over commodity soy varieties, the DNA construct of soybean plant MON87754, the detection of the transgene/genomic insertion region into soybeans creating the MON87754 event, the progeny thereof and the uses of the elevated oil provided by this invention.
Soybeans are an important crop worldwide and are a primary food source in many areas of the world. In the past, the methods and techniques of biotechnology have been applied to soybeans to improve certaineagronomic traits, and the quality of the product. According to the current invention methods have been employed to enhance the soybean oil level produced in a plant which is elevated relative to commodity soy varieties.
In the process of developing transgenic soybeans it would be advantageous to be able to detect the presence of a specific transgene in thegenomic DNA of a particular plant in order to determine whether progeny of a sexual cross contain the transgene/genomic DNA of interest. In addition, a method for detecting a particular plant with a specific transgenic insert would be helpful when complying with regulations requiring the pre-market approval and labeling of foods derived from the recombinant crop plants.
Triacylglycerol (TAG) is thought to be the most important storage unit of energy in plant cells. Diacylglycerol acyltransferase (DGAT) is an enzyme which is believed to regulate the chemical structure of TAG and to direct TAG synthesis. The reaction catalyzed by DGAT is at a critical branchpoint in glycerolipid biosynthesis. Enzymes at such branch points are considered prime candidates for sites of metabolic regulation. There are several enzymes which are common to the synthesis of diacylglycerol, TAG and membrane lipids, however the DGAT reaction is specific for oil synthesis.
In plants, TAG is the primary component of vegetable oil that is used by the seed as a stored form of energy to be used during seed germination. Higher plants appear to synthesize oils via a common metabolic pathway. Fatty acids are made in plastids from acetyl-CoA through a series of reactions catalyzed by enzymes known collectively as Fatty Acid Synthase (FAS). The fatty acids produced in plastids are exported to the cytosolic compartment of the cell, and are esterified to coenzyme A. These acyl-CoAs are the substrates for glycerolipid synthesis in the endoplasmic reticulum (ER). Glycerolipid synthesis itself is a series of reactions leading first to phosphatidic acid (PA) and Diacylglycerol (DAG). Either of these metabolic intermediates may be directed to membrane phospholipids such as phosphatidylglycerol (PG) phosphatidylethanolamine (PE) or phosphatidylcholine (PC) or they may be directed on to form neutral triacylglycerol (TAG).
Diacylglycerol (DAG) is synthesized from glycerol-3-phosphate and fatty acyl-CoAs in two steps catalyzed sequentially by glycerol-3-phosphate acyltransferase (G3PAT), and lysophosphatidic acid acyltransferase (LPAAT) to make PA, and then an additional hydrolytic step catalyzed by phosphatidic acid phosphatase (PAP) to make DAG. In most cells, DAG is used to make membrane phospholipids, the first step being the synthesis of PC catalyzed by CTP-phosphocholine cytidylyltransferase. In cells producing storage oils, DAG is acylated with a third fatty acid in a reaction catalyzed by diacylglycerol acyltransferase (DGAT). Collectively, the reactions make up part of what is commonly referred to as the Kennedy Pathway.
TAGs present in plants and animals are important molecules as energy reserve. In oilseed crops, TAG plays a major role as storage lipids and utilized as plants oil. Crude soybean oil, which is traded at international grain market as ‘soybean oil’, is composed of 95-97% triacylglycerols (TAGs), and refined soybean oil for edible use is composed of >99% TAGs (Perkins, 1995); therefore any increase in triacylglycerols leads to an increase in soybean oil content.
Soybeans are the largest source of vegetable oil worldwide (USDA, 2007). The world demand for vegetable oil grows at an accelerating rate driven by the rapid economical expansion in the emerging markets such as China and India. In recent years energy prices have risen to historical record level and will most likely stay high for the foreseeable future. This energy price surge has further stimulated demand for alternative energy sources including biodiesel, which can be produced from soybean oil. Thus with soybean oil becoming more valuable, traits that increase total soybean oil content will improve the total value of this crop.
Controlled expression of the DGAT gene in a transgenic plant to produce a seed with a higher ratio of oil to seed meal would be useful to obtain a desired oil at a lower cost. This would be typical of a high value oil product. Or such an oilseed might constitute a superior feed for animals. In some instances having an oilseed with a lower ratio of oil to seed meal would be useful to lower caloric content. In other uses, edible plant oils with a higher percentage of unsaturated fatty acids are desired for cardiovascular health reasons. And alternatively, temperate substitutes for high saturate tropical oils such as palm, coconut or cocoa would also find uses in a variety of industrial and food applications.
The expression of foreign genes in plants is known to be influenced by their chromosomal position, perhaps due to chromatin structure (e.g., heterochromatin) or the proximity of transcriptional regulation elements (e.g., enhancers) close to the integration site Weising et al. (1988 Ann. Rev. Genet 22:421-477). For this reason, it is often necessary to screen a large number of events in order to identify an event characterized by optimal expression of an introduced gene of interest. For example, it has been observed in plants and in other organisms that there may be wide variation in the levels of expression of an introduced gene among events. There may also be differences in spatial or temporal patterns of expression, for example, differences in the relative expression of a transgene in various plant tissues, that may not correspond to the patterns expected from transcriptional regulatory elements present in the introduced gene construct. For this reason, it is common to produce several hundreds to several thousands different events and screen the events for a single event that has the desired transgene expression levels and patterns for commercial purposes. An event that has the desired levels or patterns of transgene expression is useful for introgressing the transgene into other genetic backgrounds by sexual outcrossing using conventional breeding methods. Progeny of such crosses maintain the transgene expression characteristics of the original transformant. This strategy is used to ensure reliable gene expression in a number of varieties that are suitably adapted to specific local growing conditions.
It is possible to detect the presence of a transgene by any well known nucleic acid detection method such as the polymerase chain reaction (PCR) or DNA hybridization using nucleic acid probes. These detection methods generally focus on frequently used genetic elements, such as promoters, terminators, marker genes, etc. As a result, such methods may not be useful for discriminating between different events, particularly those produced using the same DNA construct unless the sequence of chromosomal DNA adjacent to the inserted DNA (“flanking DNA”) is known. An event-specific PCR assay is discussed, for example, by Taverniers et al. (J. Agric. Food Chem., 53: 3041-3052, 2005) in which an event-specific tracing system for transgenic maize lines Bt11, Bt176, and GA21 and for canola event GT73 is demonstrated. In this study, event-specific primers and probes were designed based upon the sequences of the genome/transgene junctions for each event. Transgenic plant event specific DNA detection methods have also been described in U.S. Pat. Nos. 6,893,826; 6,825,400; 6,740,488; 6,733,974; 6,689,880; 6,900,014 and 6,818,807.